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Biomedical subjects

G B Bouck

Publications and source records attributed to G B Bouck.

28 records · Page 2Linked to original sources
PubMed1974

Microtubule biogenesis and cell shape in Ochromonas. 3. Effects of herbicidal mitotic inhibitor isopropyl N-phenylcarbamate on shape and flagellum regeneration.

The role of microtubules and microtubule nucleating sites in the unicell, Ochromonas has been examined through the use of two mitotic inhibitors, isopropyl N-phenylcarbamate (IPC) and isopropyl N-3-chlorophenyl carbamate (CIPC). Although IPC and CIPC have little or no effect on intact microtubules, the assembly of three separate sets of microtubules in Ochromonas has been found to be differentially affected by IPC and CIPC. The assembly of flagellar microtubules after mechanical deflagellation is partially inhibited; the reassembly of rhizoplast microtubules after pressure depolymerization is totally inhibited (however, macrotubules may form at the sites of microtubule initiation or elsewhere); and, the reassembly of the beak set of microtubules after pressure depolymerization may be unaffected although similar concentrations of IPC and CICP completely inhibit microtubule regeneration on the rhizoplast. These effects on microtubule assembly, either inhibitory or macrotubule inducing, are fully reversible. The kinetics of inhibition and reversal are found to be generally similar for both flagellar and cell shape regeneration. Incorporation data suggest that neither IPC nor CIPC has significant effects on protein synthesis in short term experiments. Conversely, inhibiting protein synthesis with cycloheximide has little effect on microtubule regeneration when IPC or CIPC is removed. Although the exact target for IPC and CIPC action remains uncertain, the available evidence suggests that the microtubule protein pool or the microtubule nucleating sites are specifically and reversibly affected. Comparative experiments using the mitotic inhibitor colchicine indicate some similarities and differences in its mode of action with respect to that of IPC and CIPC on assembly and disassembly of microtubules in these cells.

Amino Acids↗
PubMed1973

Microtubule biogenesis and cell shape in Ochromonas. I. The distribution of cytoplasmic and mitotic microtubules.

In the first of two companion papers which attempt to correlate microtubules and their nucleating sites with developmental and cell division patterns in the unicellular flagellate, Ochromonas, the distribution of cytoplasmic and mitotic microtubules and various kinetosome-related fibers are detailed. Of the five kinetosome-related fibers, which have been found in Ochromonas, two, the kineto-beak fibers and the rhizoplast fibers are utilized as attachment sites for distinct groups of microtubules. The set of microtubules attached to the kineto-beak fibers apparently shape the anterior beak region of the cell whereas the rhizoplast microtubules appear to extend into and shape the tail in vegetative cells. In mitotic cells a rhizoplast is found at each spindle pole apparently serving as foci for the spindle microtubules. These findings are discussed in relation to the less well defined attachment sites for vegetative and mitotic microtubules in other kinds of cells. It is noted that the effects of depolymerizing microtubules in vivo might be easily quantitated in whole populations since no external wall or pellicle contributes to the maintenance or the biogenesis of the characteristic cell form of Ochromonas.

Cell Division↗
PubMed1973

Microtubule biogenesis and cell shape in Ochromonas. II. The role of nucleating sites in shape development.

The proposal made in the preceding paper that the species-specific shape of Ochromonas is mediated by cytoplasmic microtubules which are related to two nucleating sites has been experimentally verified. Exposure of cells to colchicine or hydrostatic pressure causes microtubule disassembly and a correlative loss of cell shape in a posterior to anterior direction. Upon removal of colchicine or release of pressure, cell shape regenerates and microtubules reappear, first in association with the kineto-beak site concomitant with regeneration of the anterior asymmetry, and later at the rhizoplast site concomitant with formation of the posterior tail. It is concluded that two separate sets of cytoplasmic tubules function in formation and maintenance of specific portions of the total cell shape. On the basis of the following observations, we further suggest that the beak and rhizoplast sites could exert control over the position and timing of the appearance, the orientation, and the pattern of microtubule distribution in Ochromonas. (a) the two sites are accurately positioned in the cell relative to other cell organelles; (b) in regenerating cells microtubules reform first at these sites and appear to elongate to the cell posterior; (c) microtubules initially reappear in the orientation characteristic of the fully differentiated cell; (d) the two sets of tubules are polymerized at different times, in the same sequence, during reassembly or resynthesis of the microtubular system. Experiments using cycloheximide, after a treatment with colchicine, have demonstrated that Ochromonas cannot reassume its normal shape without new protein synthesis. This suggests that microtubule protein once exposed to colchicine cannot be reassembled into microtubules. Pressure-treated cells, on the other hand, reassemble tubules and regenerate the normal shape in the presence or absence of cycloheximide. The use of these two agents in analyzing nucleating site function and the independent processes of synthesis and assembly of microtubules is discussed.

Amino Acids↗
PubMed1971

The structure, origin, isolation, and composition of the tubular mastigonemes of the Ochromas flagellum.

The structure, assembly, and composition of the extracellular hairs (mastigonemes) of Ochromonas are detailed in this report. These mastigonemes form two lateral unbalanced rows, each row on opposite sides of the long anterior flagellum. Each mastigoneme consists of lateral filaments of two distinct sizes attached to a tubular shaft. The shaft is further differentiated into a basal region at one end and a group of from one to three terminal filaments at the free end. Mastigoneme ontogeny as revealed especially in deflagellated and regenerating cells appears to begin by assembly of the basal region and shaft within the perinuclear continuum. However, addition of lateral filaments to the shaft and extrusion of the mastigonemes to the cell surface is mediated by the Golgi complex. The ultimate distribution of mastigonemes on the flagellar surface seems to be the result of extrusion of mastigonemes near the base of the flagellum, and it is suggested that mastigonemes are then pulled up the flagellum as the axoneme elongates. Efforts to characterize mastigonemes biochemically after isolation and purification on cesium chloride (CsCl) followed by electrophoresis on acrylamide gels have demonstrated what appear to be a single major polypeptide and several differentially migrating carbohydrates. The polypeptide is not homologous with microtuble protein. The functionally anomalous role of mastigonemes in reversing flagellar thrust is discussed in relation to their distribution relative to flagellar anatomy and to the plane of flagellar undulations.

Carbohydrates↗
PubMed1969

Extracellular microtubules. The origin, structure, and attachment of flagellar hairs in Fucus and Ascophyllum antherozoids.

Mastigonemes (Flimmer) from the sperm of Ascophyllum and Fucus were found to consist of a tripartite structure-a ca. 2000-A tapered basal region, a closed microtubular shaft, and a group of terminal filaments. Each of these regions appears to be constructed of globular subunits with a center-to-center distance of about 45 A. The mastigoneme microtubule is of smaller diameter (170-190 A) than cytoplasmic microtubules in these or other plant cells. During the initial stages of flagellar ontogeny, structures similar to mastigonemes (presumptive mastigonemes) are found within membrane-limited sacs in the cytoplasm or within the perinuclear space. Mastigonemes at this time are generally not found on the flagellar surface. Later, when the anterior flagellum acquires mastigonemes, the presumptive mastigonemes are absent from the cytoplasm. The regularity of attachment of mastigonemes to the flagellar surface suggests that specific attachment sites are constructed on the plasma membrane during flagellar ontogeny. No evidence for penetration of the mastigoneme through the plasma membrane was obtained. The origin and structure of mastigonemes are discussed in relation to reports of the origin and structure of other microtubular systems.

Eukaryota↗
PubMed1965

Fine structure and organelle association in brown algae.

The structural interrelationships among several membrane systems in the cells of brown algae have been examined by electron microscopy. In the brown algae the chloroplasts are surrounded by two envelopes, the outer of which in some cases is continuous with the nuclear envelope. The pyrenoid, when present, protrudes from the chloroplast, is also surrounded by the two chloroplast envelopes, and, in addition, is capped by a third dilated envelope or "pyrenoid sac." The regular apposition of the membranes around the pyrenoid contrasts with their looser appearance over the remainder of the chloroplast. The Golgi apparatus is closely associated with the nuclear envelope in all brown algae examined, but in the Fucales this association may extend to portions of the cytoplasmic endoplasmic reticulum as well. Evidence is presented for the derivation of vesicles, characteristic of those found in the formative region of the Golgi apparatus, from portions of the underlying nuclear envelope. The possibility that a structural channeling system for carbohydrate reserves and secretory precursors may be present in brown algae is considered. Other features of the brown algal cell, such as crystal-containing bodies, the variety of darkly staining vacuoles, centrioles, and mitochondria, are examined briefly, and compared with similar structures in other plant cells.

Cell Membrane↗
PubMedDate not supplied

Tubulin genes in the algal protist Euglena gracilis.

Alpha- and beta-tubulin cDNA were selected from a Euglena lambda gt11 expression library, recloned and either sequenced (alpha-tubulin cDNA) or hybridized to Euglena RNA and DNA (alpha- and beta-tubulin cDNA). RNA for hybridization was extracted at 30 minute intervals after flagellar amputation and quantitated for cDNA binding. Unlike previous reports on most other flagellates, no net increase in either alpha- or beta-tubulin RNA could be detected during regeneration--suggesting steady state or constitutive tubulin RNA synthesis. Incubation of the cDNA with genomic DNA after restriction digestion produced patterns of hybridization consistent with the presence of one to two kinds each of the alpha- and beta-tubulin genes. The deduced amino acid sequence of the alpha-tubulin cDNA was more than 90% identical to the alpha-tubulins of Trypanosoma, Chlamydomonas, Naegleria, Tetrahymena and higher plants. The carboxy terminus of the alpha-tubulin cDNA and the previously sequenced beta-tubulin of Euglena showed greatest identity to the carboxy terminus of the tubulins from Trypanosoma brucei. The sequence data for alpha- and beta-tubulins of Euglena provides direct evidence for the similarity of two gene products from euglenas and trypanosomes and adds support to earlier suggestions that these organisms are phylogenetically related.

Amino Acid Sequence↗
PubMedDate not supplied

Membrane skeletal proteins and their integral membrane protein anchors are targets for tyrosine and threonine kinases in Euglena.

Proteins of the membrane skeleton of Euglena gracilis were extensively phosphorylated in vivo and in vitro after incubation with [32P]-orthophosphate or gamma-[32P] ATP. Endogenous protein threonine/serine activity phosphorylated the major membrane skeletal proteins (articulins) and the putative integral membrane protein (IP39) anchor for articulins. The latter was also the major target for endogenous protein tyrosine kinase activity. A cytoplasmic domain of IP39 was specifically phosphorylated, and removal of this domain with papain eliminated the radiolabeled phosphoamino acids and eliminated or radically shifted the PI of the multiple isoforms of IP39. In gel kinase assays IP39 autophosphorylated and a 25 kDa protein which does not autophosphorylate was identified as a threonine/serine (casein) kinase. Plasma membranes from the membrane skeletal protein complex contained threonine/serine (casein) kinase activity, and cross-linking experiments suggested that IP39 was the likely source for this membrane activity. pH optima, cation requirements and heparin sensitivity of the detergent solubilized membrane activity were determined. Together these results suggest that protein kinases may be important modulators of protein assembly and function of the membrane skeleton of these protistan cells.

Adenosine Triphosphate↗
PubMedDate not supplied

Heterogeneity and a coiled coil prediction of trypanosomatid-like flagellar rod proteins in Euglena.

The emergent flagellum of euglenoids and trypanosomatids contained in addition to microtubules a prominent filamentous structure--the flagellar rod (paraflagellar/paraxonemal rod). Immunoblots and immunofluorescence localization using three antibodies generated against gel-isolated proteins confirmed previous studies that the Euglena flagellar rod consisted of polypeptides migrating at 66-, 69-, and 75-kD. Immunoblotting after two dimensional gel electrophoresis identified ten or more isoforms of these polypeptides. Differences in migration in acrylamide gels under nonreducing and reducing conditions suggested that the rod proteins contain intramolecular disulfide linkages. Comparative peptide mapping showed that the 66-, 69-, and 75-kD polypeptides were distinct, but related proteins, and also identified a fourth related protein migrating at 64-kD. Using antibodies against rod proteins, two overlapping cDNAs were isolated and from their sequences the cDNAs were predicted to encode 334 amino acids of the 66-kD protein; the amino acid sequence had > 65% identity to the carboxyl-terminus of the trypanosomatid flagellar rod proteins. Secondary structural prediction suggested that flagellar rod proteins contain an extended segmented coiled coil stalk and two nonhelical heads. Coiled coil appeared to be an important structural motif in the construction of flagellar rod filaments.

Amino Acid Sequence↗